In the field of immunohistochemistry, IHC, biological targets of interest are typically stained with enzymatically generated dyes. However, most of today's IHC enzymatic systems have a limited usability of for target visualization due to restricted sensitivity: if a target is of very low abundance, the amount of deposited dye remains undetectable. Likewise, there is an upper detection limit above which a further dye deposition does not lead to detectably more intense stains. Using lower concentration of reagents, the upper detection limit may be compromised to allow differentiation between high and very high abundance targets; however this also leads to an increase of the lower detection limit, i.e. the loss in sensitivity of detection. Thus, most of the today's systems have a limited dynamic range of detection. Further, differences in sensitivity between different visualization systems from same or different vendors makes comparison the staining results difficult.
A further challenge is quantification of immunochemically stained targets due to the dye deposition is not a linear function of target concentration. Around the baseline of detection limit the intensity increases rapidly as a direct function of target concentration (as the going from no detectable signal to a signal, even of a low strength, represents an infinite increase. Conversely, close to the upper detection limit, even a large increase in target concentration will lead to virtually no perceptible increase in the already intense signal.
A further complication arises from the fact that no internationally recognized standards exist, and invariable reference samples are difficult to prepare. Even serial sections of the same tissue sample usually exhibit biological variation. Immortal cell lines might in principle provide the infinite reference material, however differences in cultivation conditions, cell cycle circles and biological variation will also in this case lead to some batch to batch variation in target expression. Glass slides chemically modified with peptides or proteins may be used as surrogate targets, however comparison to tissue samples is not straight forwards.
Thus there is a need for standardized quantitative detection of immobilized targets in biological samples.
Recently described methods of immunochemical staining of immobilized targets in biological samples, including histological samples, (WO2010094284, WO2010094283, WO201047680, WO2012143010) provide a visualization system characterized by an extreme sensitivity, such as single units of the target can be visualized and detected, which also allows a precise quantification of the target (as described in WO2012075028 WO2012062318), as the amount of deposited dye is in a linear correlation with the target expression. However, the latter visualization methods due to their extreme sensitivity, may in some cases have disadvantages, e.g. the methods are best applicable for visualization of a sub-fraction of a target in the sample, but not the whole expression range. This disadvantage may be an obstacle for broad use of the methods for visualization of targets in histological samples, especially when robustness of the procedure and reproducibility of the results are concerned. Further, the precise quantification of a target according to the methods may be laborious. The present invention solves the latter mentioned problems.